Cholecystokinin (CCK) is a neuropeptide which display many properties of a neurotransmitter. It is thought to play an important role in the neurochemical balance of the brain and alterations in its expression or secretion may play a role in Neurological and Psychiatric Disease. Very little is known about the mechanism and regulation of CCK biosynthesis. An enzyme which generates CCK8 from CCK 33 (CCK 8 generating enzyme) has been isolated from rate brain synaptosome and cDNA clones have been identified by expression cloning which have identical properties to the isolated enzyme. Since CCK 8 is the major product of pro-CCK processing in the brain, this enzyme may be the rate limiting enzyme in the biosynthesis of CCK. A number of dibasic processing enzymes (PC1 and PC2) which have been cloned by other investigators may play a role in the processing of pro-CCK at dibasic sites. The following specific aims are designed to study in detail the possible role of CGE, PC1 and PC2 in the processing of pro-CCK: 1. Isolation and sequencing of full length CGE cDNA clones. 2. The distribution an molecular forms of CGE will be examined in rat brain, pituitary, intestine, and CCK-secreting tumor cells in culture using in situ hybridization, Northern analysis, radioimmunoassay, Western immunoblot analysis and immunocytochemistry. The possible co-localization of CGE and CCK will be examined by dual immunofluorescent staining. 3. The ability of CGE to cleave recombinant pro-CCK in vitro at monobasic and dibasic sites will be evaluated using both purified CGE produced by recombinant techniques. The ability of CGE, PC1 and PC2 to process pro- CCK in living cells will be evaluated by co-expressing them with pro-CCK in CV1 cells and analyzing the products. 4. The possibility that expression of CGE, PC1, PC2 and CCK mRNA are coordinately regulated in CCK-secreting tumor cells will be examined using Northern analysis. 5. The effect of inhibition of enzyme expression on pro-CCK processing will be evaluated in CCK-secreting tumor cells using CGE, PC1, and PC2 antisense cDNAs to block the expression of their mRNAs.